Functional Effects of Glycosylation at Asn-579 of the Epidermal Growth Factor Receptor

Abstract

We have investigated functional effects of glycosylation at N(579) of the epidermal growth factor receptor (EGFR). Our previous study showed that the population of cell-surface expressed EGFRs in A431 cells, a human epidermoid carcinoma cell line, is composed of two subpopulations that differ by glycosylation at N(579) [Zhen et al. (2003) Biochemistry 42, 5478-5492]. To characterize the subpopulation of receptors not glycosylated at N(579), we established a 32D cell line expressing a point mutant of the EGFR (N579Q), which cannot be glycosylated at this position. Analysis of epitope accessibility suggests that the lack of glycosylation at N(579) weakens auto-inhibitory tether interactions, and cross-linking experiments suggest a somewhat elevated level of preformed N579Q-EGFR dimers in the absence of ligand relative to wild-type EGFR (WT-EGFR). However, ligand drives the majority of N579Q-EGFR dimerization, suggesting that untethering, while necessary, is not sufficient to drive dimerization. Ligand-binding experiments reveal a much greater fraction of N579Q-EGFRs in a high-affinity state compared to the fraction of WT-EGFRs in a high-affinity state. However, differences in the kinetic association and dissociation rates indicate that the high-affinity states of the WT and the N579Q receptors are distinct. EGF-stimulated phosphorylation in cells expressing N579Q-EGFRs results in notable differences in the pattern of tyrosine phosphorylated proteins compared with that obtained in cells expressing WT-EGFRs. Moreover, although WT-EGFRs confer cell survival in 32D cells in the absence of interleukin-3 and EGF, we found that receptors lacking glycosylation at N(579) do not. This is the first study of which we are aware to show that selective glycosylation of a specific N-glycosylation site can produce two functionally distinct receptors.

Human Epidermal Growth Factor Receptor (EGFR) Aligned on the Plasma Membrane Adopts Key Features of Drosophila EGFR Asymmetry

“…It is tempting to speculate that the flat configuration may be stabilized via interactions between hEGFR and other membrane components, including other membrane proteins, such as sugar-mediated interactions with gangliosides in the upper leaflet of the membrane (24,44), consistent with prior observations that high-affinity EGF binding to EGFR is not seen outside cell membranes (22) and that altering the glycosylation of EGFRs in cells produces altered equilibrium binding kinetics (51). Yet intracellular interactions must also be involved in promoting the flat configuration, despite the apparent loose coupling between the extracellular and intracellular regions (28), because hEGFR mutants with deleted intracellular regions do not show negative cooperativity (25,30) but have extracellular regions that are presumably competent to interact with other membrane components.…”

The Asn418-Linked N-Glycan of ErbB3 Plays a Crucial Role in Preventing Spontaneous Heterodimerization and Tumor Promotion

“…The issue of whether the conformations of spontaneous dimerization of N420Q mutant of EGFR (19) or N418Q mutant of ErbB3 are similar to that of ligand-induced dimerization is not known at present; however, the oligosaccharide on Asn 420 of EGFR or Asn 418 of ErbB3 might be involved in tethering, and thus, deleting those oligosaccharides would result in spontaneous dimerization (31). It has recently been suggested that the glycosylation at Asn 579 of EGFR plays a role in ligand binding and dimerization (32); the dimerization level of N579Q-EGFR is elevated compared with wild-type EGFR, and it has been suggested that the N-glycan on N 579 , situated at the tip of the subdomain IV autoinhibitory tether loop, serves to strengthen the tether. The addition of an N-glycan may alter the asparagine side-chain torsion angle distribution and reduces its flexibility and may be involved in stabilizing the protein folding (33).…”

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

“…ab231). Therapeutic EGFR a large N-linked carbohydrate is found on residue N328 in very close proximity to the presumed binding site for dIII/A antibodies, which together with N-linked glycans on dI 33 could limit the flexibility of dI/II and dIII/A antibodies. Finally, antibodies binding to epitopes on dI, dII and dIV are all predicted to be closer to the cell membrane than dIII antibodies and hence sterically more constrained and less able to cross-link receptors, which may explain the non-synergistic activity of such antibodies in combinations.…”